A three-way interface of the Nipah virus phosphoprotein X-domain coordinates polymerase movement along the viral genome.

Nipah virus (NiV) is a highly pathogenic paramyxovirus causing frequently lethal encephalitis in humans. The NiV genome is encapsidated by the nucleocapsid (N) protein. RNA synthesis is mediated by the viral RNA-dependent RNA polymerase (RdRP), consisting of the polymerase (L) protein complexed with the homo-tetrameric phosphoprotein (P). The advance of the polymerase along its template requires iterative dissolution and reformation of transient interactions between P and N protomers in a highly regulated process that remains poorly understood. This study applied functional and biochemical NiV polymerase assays to the problem. We mapped three distinct protein interfaces on the C-terminal P-X domain (P-XD), which form a triangular prism and engage L, the C-terminal N tail, and the globular N core, respectively. Transcomplementation assays using NiV L and N-tail binding-deficient mutants revealed that only one XD of a P tetramer binds to L, whereas three must be available for N-binding for efficient polymerase activity. The dissolution of the N-tail complex with P-XD was coordinated by a transient interaction between N-core and the α-1/2 face of this XD but not unoccupied XDs of the P tetramer, creating a timer for coordinated polymerase advance. IMPORTANCE: Mononegaviruses comprise major human pathogens such as the Ebola virus, rabies virus, respiratory syncytial virus, measles virus, and Nipah virus (NiV). For replication and transcription, their polymerase complexes must negotiate a protein-encapsidated RNA genome, which requires the highly coordinated continuous formation and resolution of protein-protein interfaces as the polymerase advances along the template. The viral P protein assumes a central role in this process, but the molecular mechanism of ensuring polymerase mobility is poorly understood. Studying NiV polymerase complexes, we applied functional and biochemical assays to map three distinct interfaces in the NiV P XD and identified transient interactions between XD and the nucleocapsid core as instrumental in coordinating polymerase advance. These results define a conserved molecular principle regulating paramyxovirus polymerase dynamics and illuminate a promising druggable target for the structure-guided development of broad-spectrum polymerase inhibitors.